A recognized problem in recombinant protein production is the toxicity of foreign proteins to the recombinant host. Desirably, the protein should be produced in large amounts-as much as about 40% of the total production of protein in the cell. However, foreign proteins are inherently aberrant in the host and therefore are often unhealthy for the cells at high concentration levels, even if they are not toxins in the conventional sense.
One approach to mitigating this problem is to delay the production of the desired protein until the cells have achieved a satisfactory level of growth. While the enhanced production at that time may eventually have a morbidity effect on the culture, there are sufficient cells available by the time protein production is induced to supply sufficient yield before morbidity occurs. To effect this delay, conventionally, inducible promoter systems have been employed. In mammalian cells the metallothionein promoter has been most commonly used. "Superinduction" protocols have been devised involving treatment with zinc ion which give a 50-fold induction of the synthesis of human leutropin (Walden, W. E., et al., Gene (1987) 61:317-327) or the encephalomyocarditis (EMC) virus 3B and 3C proteins (Lawson, T. G., et al., J. Virol. (1989) 63:5013-5022). Considerable cell lysis resulted from these enhanced concentrations of protein. As very high levels of protein production were achieved, an increase in overall yield is achievable only by enhancing the effectiveness of induction. This comprises suppressing both transcription and translation of the transcripts so as to minimize cytotoxicity during clonal selection and cell proliferation phases to enhance the probability of isolating cell lines that can be induced to produce large quantities of the desired protein.
In addition to regulating the production of the protein at the transcriptional level, regulation at the translational level is also known. The mRNA transcript of all ferritins contains a sequence of about 27 to about 60 nucleotides in the 5'-untranslated region which appears to repress translation of the mRNA in the absence of iron; when a source of iron ion is added, translation can go forward. This "iron-responsive element" (IRE) is thought to form a stem-loop structure which is recognized by ferritin repressor protein or "IRE-binding" protein. The function of the iron ion appears to reside in its ability to inactivate the repressor protein (Hentze, M. W., et al., Science (1987) 238:1570-1573; Aziz, N., and Munro, H. N., Proc. Natl. Acad. Sci. USA (1987) 84:8478- 8482; Brown, P. H., et al., J. Biol. Chem. (1989) 264:13383-13386).
While the ability of the IRE to control the translation of ferritin so as to permit controlled protein production is established, this translation regulator does not seem to be effective with respect to all coding sequences contained in the mRNA. It has now been found that the combination of an inducible translation regulator such as IRE with a message-masking element (MME) results in a dependable translation regulation system which permits control of recombinant protein expression. This combination, when further used in conjunction with a transcription regulator, results in an approximately 500-fold induction for most proteins.